Zazali Alias

Binding of an anticancer drug, axitinib to human serum albumin: Fluorescence quenching and molecular docking study.

Binding characteristics of a promising anticancer drug, axitinib (AXT) to human serum albumin (HSA), the major transport protein in human blood circulation, were studied using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as molecular docking analysis. A gradual decrease in the Stern-Volmer quenching constant with increasing temperature revealed the static mode of the protein fluorescence quenching upon AXT addition, thus confirmed AXT-HSA complex formation. This was also confirmed from alteration in the UV-vis spectrum of HSA upon AXT addition. Fluorescence quenching titration results demonstrated moderately strong binding affinity between AXT and HSA based on the binding constant value (1.08±0.06×10(5)M(-1)), obtained in 10mM sodium phosphate buffer, pH7.4 at 25°C. The sign and magnitude of the enthalpy change (∆H=-8.38kJmol(-1)) as well as the entropy change (∆S=+68.21Jmol(-1)K(-1)) clearly suggested involvement of both hydrophobic interactions and hydrogen bonding in AXT-HSA complex formation. These results were well supported by molecular docking results. Three-dimensional fluorescence spectral results indicated significant microenvironmental changes around Trp and Tyr residues of HSA upon complexation with AXT. AXT binding to the protein produced significant alterations in both secondary and tertiary structures of HSA, as revealed from the far-UV and the near-UV CD spectral results. Competitive drug displacement results obtained with phenylbutazone (site I marker), ketoprofen (site II marker) and hemin (site III marker) along with molecular docking results suggested Sudlows site I, located in subdomain IIA of HSA, as the preferred binding site of AXT.

Interaction of an anticancer drug, gefitinib with human serum albumin: insights from fluorescence spectroscopy and computational modeling analysis

Binding of gefitinib (GEF), a promising anticancer drug to human serum albumin (HSA), the major transport protein in blood circulation was investigated using fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy as well as computational modeling. Fluorescence quenching of HSA upon GEF addition was found to be a static quenching process, as revealed from the decreasing trend of the Stern–Volmer quenching constant with increasing temperature as well as UV-vis absorption spectral results. Fluorescence quenching titration results demonstrated moderate binding affinity with the binding constant, Ka value as 1.70 × 104 M−1 between GEF and HSA at 15 °C. Thermodynamic data (ΔH = −7.74 kJ mol−1 and ΔS = +54.06 J mol−1 K−1) suggested participation of both hydrophobic interactions and hydrogen bonds in stabilizing the GEF–HSA complex, which was further supported by computational modeling results. The far-UV and the near-UV CD spectra showed secondary and tertiary structural changes in HSA, whereas three-dimensional fluorescence spectral results indicated microenvironmental perturbations around protein fluorophores upon GEF binding. Binding of GEF to HSA offered significant protection to the protein against thermal destabilization. Competitive site-marker displacement results along with computational modeling analysis suggested a preferred location of the GEF binding site as site III, located in subdomain IB of HSA. Some common metal ions have been found to interfere with GEF–HSA interaction.

Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking

Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89 × 103 M−1 at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol−1 K−1) and negative ΔH (−6.57 kJ mol−1) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca2+, Zn2+, Cu2+, Ba2+, Mg2+, and Mn2+ in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA. Graphical abstract Binding orientation of the VDB in the Sudlow’s binding site I (subdomain IIA) of HSA.

Characterization of the binding of an anticancer drug, lapatinib to human serum albumin.

Interaction of a promising anticancer drug, lapatinib (LAP) with the major transport protein in human blood circulation, human serum albumin (HSA) was investigated using fluorescence and circular dichroism (CD) spectroscopy as well as molecular docking analysis. LAP-HSA complex formation was evident from the involvement of static quenching mechanism, as revealed by the fluorescence quenching data analysis. The binding constant, Ka value in the range of 1.49-1.01×10(5)M(-1), obtained at three different temperatures was suggestive of the intermediate binding affinity between LAP and HSA. Thermodynamic analysis of the binding data (∆H=-9.75kJmol(-1) and ∆S=+65.21Jmol(-1)K(-1)) suggested involvement of both hydrophobic interactions and hydrogen bonding in LAP-HSA interaction, which were in line with the molecular docking results. LAP binding to HSA led to the secondary and the tertiary structural alterations in the protein as evident from the far-UV and the near-UV CD spectral analysis, respectively. Microenvironmental perturbation around Trp and Tyr residues in HSA upon LAP binding was confirmed from the three-dimensional fluorescence spectral results. LAP binding to HSA improved the thermal stability of the protein. LAP was found to bind preferentially to the site III in subdomain IB on HSA, as probed by the competitive drug displacement results and supported by the molecular docking results. The effect of metal ions on the binding constant between LAP and HSA was also investigated and the results showed a decrease in the binding constant in the presence of these metal ions.

Comprehensive insight into the binding of sunitinib, a multi-targeted anticancer drug to human serum albumin

Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04×104M-1), obtained at 298K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlows site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.

Biophysical and computational characterization of vandetanib–lysozyme interaction

Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased proteins thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.

Effects and optimization of selected operating variables on laccase production from Pycnoporus sanguineus in strirred tank reactor.

Abstract The effects of selected variables in the production of laccase by Pycnoporus sanguineus using 2 L stirred tank reactor were investigated. Three operating variables namely agitation rate, aeration rate and pH were studied and optimized for maximum enzyme production. Screening experiments of the three variables were done using 2 level full factorial design (FFD). Analysis on factorial screening indicated laccase production was significantly influenced by agitation rate and pH and not aeration rate. The effect of agitation and pH were further optimized using face centered central composite (FCCCD) with fixed aeration rate. The initial factorial screening was augmented with new experiments in order to produce face-centered design for the optimization. Statistical analysis indicated the experimental data fitted well a quadratic model. Model validation was done through residual analysis. Maximum laccase production of 67.1 U L-1 was predicted and experimentally validated under the proposed optimum conditions.

Fishmeal replacement with Spirulina Platensis and Chlorella vulgaris in African catfish ( Clarias gariepinus ) diet: Effect on antioxidant enzyme activities and haematological parameters

This study explored fishmeal replacement with two freshwater microalgae: Spirulina Platensis and Chlorella vulgaris in African catfish (Clarias gariepinus) diet. The effect of inclusion of the two microalgae on biomarkers of oxidative stress, haematological parameters, enzyme activities and growth performance were investigated. The juvenile fish were given 3 distinct treatments with isonitrogenous (35.01-36.57%) and isoenergetic (417.24-422.27 Kcal 100 g-1) diets containing 50% S. platensis (50SP), 75% S. platensis (75SP), 50% C. vulgaris (50CL), 75% C. vulgaris (75CL) and 100% fishmeal (100% FM) was used as the control diet. The result shows that all the diets substituted with both S. platensis, and C. vulgaris boosted the growth performance based on specific growth rate (SGR) and body weight gain (BDWG) when compared with the control diet. The feed conversion ratio (FCR) and protein efficiency ratio (PER) was significantly influenced by all the supplementations. The haematological analysis of the fish shows a significant increase in the value of red and white blood cells upon supplementation with 50SP and 50CL but decrease slightly when increased to 75SP and 75CL. Furthermore, the value of haematocrit and haemoglobin also increased upon supplementation with 50SP and 50CL but decrease slightly when increased to 75SP and 75CL. The white blood cell (WBC), red blood cell (RBC) increased, while total cholesterol (TCL), and Plasma glucose levels decreased significantly upon supplementation of algae. This is a clear indication that S. platensis and C. vulgaris are a promising replacement for fishmeal, which is a source protein in the C. gariepinus diet.

Functional Role of Tyr12 in the Catalytic Activity of Novel Zeta-like Glutathione S -transferase from Acidovorax sp. KKS102

Glutathione S-transferases (GSTs) are a family of enzymes that function in the detoxification of variety of electrophilic substrates. In the present work, we report a novel zeta-like GST (designated as KKSG9) from the biphenyl/polychlorobiphenyl degrading organism Acidovorax sp. KKS102. KKSG9 possessed low sequence similarity but similar biochemical properties to zeta class GSTs. Functional analysis showed that the enzyme exhibits wider substrate specificity compared to most zeta class GSTs by reacting with 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzyl chloride (NBC), ethacrynic acid (EA), hydrogen peroxide, and cumene hydroperoxide. The enzyme also displayed dehalogenation function against dichloroacetate, permethrin, and dieldrin. The functional role of Tyr12 was also investigated by site-directed mutagenesis. The mutant (Y12C) displayed low catalytic activity and dehalogenation function against all the substrates when compared with the wild type. Kinetic analysis using NBC and GSH as substrates showed that the mutant (Y12C) displayed a higher affinity for NBC when compared with the wild type, however, no significant change in GSH affinity was observed. These findings suggest that the presence of tyrosine residue in the motif might represent an evolutionary trend toward improving the catalytic activity of the enzyme. The enzyme as well could be useful in the bioremediation of various types of organochlorine pollutants.

Deoxyelephantopin ameliorates lipopolysaccharides (LPS)-induced memory impairments in rats: Evidence for its anti-neuroinflammatory properties

Aim: Neuroinflammation is a critical pathogenic mechanism of most neurodegenerative disorders especially, Alzheimers disease (AD). Lipopolysaccharides (LPS) are known to induce neuroinflammation which is evident from significant upsurge of pro‐inflammatory mediators in in vitro BV‐2 microglial cells and in vivo animal models. In present study, we investigated anti‐neuroinflammatory properties of deoxyelephantopin (DET) isolated from Elephantopus scaber in LPS‐induced neuroinflammatory rat model. Materials and methods: In this study, DET (0.625. 1.25 and 2.5 mg/kg, i.p.) was administered in rats for 21 days and those animals were challenged with single injection of LPS (250 &mgr;g/kg, i.p.) for 7 days. Cognitive and behavioral assessment was carried out for 7 days followed by molecular assessment on brain hippocampus. Statistical significance was analyzed with one‐way analysis of variance followed by Dunnetts test to compare the treatment groups with the control group. Key findings: DET ameliorated LPS‐induced neuroinflammation by suppressing major pro‐inflammatory mediators such as iNOS and COX‐2. Furthermore, DET enhanced the anti‐inflammatory cytokines and concomitantly suppressed the pro‐inflammatory cytokines and chemokine production. DET treatment also reversed LPS‐induced behavioral and memory deficits and attenuated LPS‐induced elevation of the expression of AD markers. DET improved synaptic‐functionality via enhancing the activity of pre‐ and post‐synaptic markers, like PSD‐95 and SYP. DET also prevented LPS‐induced apoptotic neurodegeneration via inhibition of PARP‐1, caspase‐3 and cleaved caspase‐3. Significance: Overall, our studies suggest DET can prevent neuroinflammation‐associated memory impairment and neurodegeneration and it could be developed as a therapeutic agent for the treatment of neuroinflammation‐mediated and neurodegenerative disorders, such as AD. Graphical abstract Figure. No caption available.